1H, 15N, and 13C chemical shift assignments of neuronal calcium sensor-1 homolog from fission yeast

The neuronal calcium sensor (NCS) proteins regulate signal transduction processes and are highly conserved from yeast to humans. We report complete NMR chemical shift assignments of the NCS homolog from fission yeast (Schizosaccharomyces pombe), referred to in this study as Ncs1p. (BMRB no. 16446)

Transient Receptor Potential (TRP) and Cch1-Yam8 Channels Play Key Roles in the Regulation of Cytoplasmic Ca2+ in Fission Yeast

The regulation of cytoplasmic Ca2+ is crucial for various cellular processes. Here, we examined the cytoplasmic Ca2+ levels in living fission yeast cells by a highly sensitive bioluminescence resonance energy transfer-based assay using GFP-aequorin fusion protein linked by 19 amino acid. We monitored the cytoplasmic Ca2+ level and its change caused by extracellular stimulants such as CaCl2 or NaCl plus FK506 (calcineurin inhibitor). We found that the extracellularly added Ca2+ caused a dose-dependent increase in the cytoplasmic Ca2+ level and resulted in a burst-like peak. The overexpression of two transient receptor potential (TRP) channel homologues, Trp1322 or Pkd2, markedly enhanced this response. Interestingly, the burst-like peak upon TRP overexpression was completely abolished by gene deletion of calcineurin and was dramatically decreased by gene deletion of Prz1, a downstream transcription factor activated by calcineurin. Furthermore, 1 hour treatment with FK506 failed to suppress the burst-like peak. These results suggest that the burst-like Ca2+ peak is dependent on the transcriptional activity of Prz1, but not on the direct TRP dephosphorylation. We also found that extracellularly added NaCl plus FK506 caused a synergistic cytosolic Ca2+ increase that is dependent on the inhibition of calcineurin activity, but not on the inhibition of Prz1. The synergistic Ca2+ increase is abolished by the addition of the Ca2+ chelator BAPTA into the media, and is also abolished by deletion of the gene encoding a subunit of the Cch1-Yam8 Ca2+ channel complex, indicating that the synergistic increase is caused by the Ca2+ influx from the extracellular medium via the Cch1-Yam8 complex. Furthermore, deletion of Pmk1 MAPK abolished the Ca2+ influx, and overexpression of the constitutively active Pek1 MAPKK enhanced the influx. These results suggest that Pmk1 MAPK and calcineurin positively and negatively regulate the Cch1-Yam8 complex, respectively, via modulating the balance between phosphorylation and dyphosphorylation state

Biochemical quantitation of the eIF5A hypusination in Arabidopsis thaliana uncovers ABA-dependent regulation

The eukaryotic translation elongation factor eIF5A is the only protein known to contain the unusual amino acid hypusine which is essential for its biological activity. This post-translational modification is achieved by the sequential action of the enzymes deoxyhypusine synthase (DHS) and deoxyhypusine hydroxylase (DOHH). The crucial molecular function of eIF5A during translation has been recently elucidated in yeast and it is expected to be fully conserved in every eukaryotic cell, however the functional description of this pathway in plants is still sparse. The genetic approaches with transgenic plants for either eIF5A overexpression or antisense have revealed some activities related to the control of cell death processes but the molecular details remain to be characterized. One important aspect of fully understanding this pathway is the biochemical description of the hypusine modification system. Here we have used recombinant eIF5A proteins either modified by hypusination or non-modified to establish a bi-dimensional electrophoresis (2D-E) profile for the three eIF5A protein isoforms and their hypusinated or unmodified proteoforms present in Arabidopsis thaliana. The combined use of the recombinant 2D-E profile together with 2D-E/western blot analysis from whole plant extracts has provided a quantitative approach to measure the hypusination status of eIF5A. We have used this information to demonstrate that treatment with the hormone abscisic acid produces an alteration of the hypusine modification system in Arabidopsis thaliana. Overall this study presents the first biochemical description of the post-translational modification of eIF5A by hypusination which will be functionally relevant for future studies related to the characterization of this pathway in Arabidopsis thaliana.Belda Palazón, B.; Nohales Zafra, MA.; Rambla Nebot, JL.; Aceña, JL.; Delgado, O.; Fustero Lardies, S.; Martínez, MC.... (2014). Biochemical quantitation of the eIF5A hypusination in Arabidopsis thaliana uncovers ABA-dependent regulation. Frontiers in Plant Science. 5:202-1-202-11. doi:10.3389/fpls.2014.00202S202-1202-115Belda-Palazón, B., Ruiz, L., Martí, E., Tárraga, S., Tiburcio, A. F., Culiáñez, F., … Ferrando, A. (2012). Aminopropyltransferases Involved in Polyamine Biosynthesis Localize Preferentially in the Nucleus of Plant Cells. PLoS ONE, 7(10), e46907. doi:10.1371/journal.pone.0046907Bergeron, R. J., Weimar, W. R., Müller, R., Zimmerman, C. O., McCosar, B. H., Yao, H., & Smith, R. E. (1998). Synthesis of Reagents for the Construction of Hypusine and Deoxyhypusine Peptides and Their Application as Peptidic Antigens. Journal of Medicinal Chemistry, 41(20), 3888-3900. doi:10.1021/jm980389pChattopadhyay, M. K., Park, M. H., & Tabor, H. (2008). Hypusine modification for growth is the major function of spermidine in Saccharomyces cerevisiae polyamine auxotrophs grown in limiting spermidine. Proceedings of the National Academy of Sciences, 105(18), 6554-6559. doi:10.1073/pnas.0710970105Chevallet, M., Luche, S., & Rabilloud, T. (2006). Silver staining of proteins in polyacrylamide gels. Nature Protocols, 1(4), 1852-1858. doi:10.1038/nprot.2006.288Cuevas, J. C., López-Cobollo, R., Alcázar, R., Zarza, X., Koncz, C., Altabella, T., … Ferrando, A. (2008). Putrescine Is Involved in Arabidopsis Freezing Tolerance and Cold Acclimation by Regulating Abscisic Acid Levels in Response to Low Temperature. Plant Physiology, 148(2), 1094-1105. doi:10.1104/pp.108.122945Czechowski, T., Stitt, M., Altmann, T., Udvardi, M. K., & Scheible, W.-R. (2005). Genome-Wide Identification and Testing of Superior Reference Genes for Transcript Normalization in Arabidopsis. Plant Physiology, 139(1), 5-17. doi:10.1104/pp.105.063743Dias, C. A. O., Garcia, W., Zanelli, C. F., & Valentini, S. R. (2012). eIF5A dimerizes not only in vitro but also in vivo and its molecular envelope is similar to the EF-P monomer. Amino Acids, 44(2), 631-644. doi:10.1007/s00726-012-1387-7Doerfel, L. K., Wohlgemuth, I., Kothe, C., Peske, F., Urlaub, H., & Rodnina, M. V. (2012). EF-P Is Essential for Rapid Synthesis of Proteins Containing Consecutive Proline Residues. Science, 339(6115), 85-88. doi:10.1126/science.1229017Duguay, J., Jamal, S., Liu, Z., Wang, T.-W., & Thompson, J. E. (2007). Leaf-specific suppression of deoxyhypusine synthase in Arabidopsis thaliana enhances growth without negative pleiotropic effects. Journal of Plant Physiology, 164(4), 408-420. doi:10.1016/j.jplph.2006.02.001Feng, H., Chen, Q., Feng, J., Zhang, J., Yang, X., & Zuo, J. (2007). Functional Characterization of the Arabidopsis Eukaryotic Translation Initiation Factor 5A-2 That Plays a Crucial Role in Plant Growth and Development by Regulating Cell Division, Cell Growth, and Cell Death. Plant Physiology, 144(3), 1531-1545. doi:10.1104/pp.107.098079Gregio, A. P. B., Cano, V. P. S., Avaca, J. S., Valentini, S. R., & Zanelli, C. F. (2009). eIF5A has a function in the elongation step of translation in yeast. Biochemical and Biophysical Research Communications, 380(4), 785-790. doi:10.1016/j.bbrc.2009.01.148Guo, J., Wang, S., Valerius, O., Hall, H., Zeng, Q., Li, J.-F., … Chen, J.-G. (2010). Involvement of Arabidopsis RACK1 in Protein Translation and Its Regulation by Abscisic Acid. Plant Physiology, 155(1), 370-383. doi:10.1104/pp.110.160663Gutierrez, E., Shin, B.-S., Woolstenhulme, C. J., Kim, J.-R., Saini, P., Buskirk, A. R., & Dever, T. E. (2013). eIF5A Promotes Translation of Polyproline Motifs. Molecular Cell, 51(1), 35-45. doi:10.1016/j.molcel.2013.04.021Hamasaki-Katagiri, N., Tabor, C. W., & Tabor, H. (1997). Spermidine biosynthesis in Saccharomyces cerevisiae: Polyaminerequirement of a null mutant of the SPE3 gene (spermidine synthase). Gene, 187(1), 35-43. doi:10.1016/s0378-1119(96)00660-9Imai, A., Matsuyama, T., Hanzawa, Y., Akiyama, T., Tamaoki, M., Saji, H., … Takahashi, T. (2004). Spermidine Synthase Genes Are Essential for Survival of Arabidopsis. Plant Physiology, 135(3), 1565-1573. doi:10.1104/pp.104.041699Ishfaq, M., Maeta, K., Maeda, S., Natsume, T., Ito, A., & Yoshida, M. (2012). Acetylation regulates subcellular localization of eukaryotic translation initiation factor 5A (eIF5A). FEBS Letters, 586(19), 3236-3241. doi:10.1016/j.febslet.2012.06.042Jin, B.-F., He, K., Wang, H.-X., Wang, J., Zhou, T., Lan, Y., … Zhang, X.-M. (2003). Proteomic analysis of ubiquitin-proteasome effects: insight into the function of eukaryotic initiation factor 5A. Oncogene, 22(31), 4819-4830. doi:10.1038/sj.onc.1206738Kang, K. R., & Chung, S. I. (2003). Protein kinase CK2 phosphorylates and interacts with deoxyhypusine synthase in HeLa cells. Experimental & Molecular Medicine, 35(6), 556-564. doi:10.1038/emm.2003.73Klier, H., Csonga, R., Joao, H. C., Eckerskorn, C., Auer, M., Lottspeich, F., & Eder, J. (1995). Isolation and Structural Characterization of Different Isoforms of the Hypusine-Containing Protein eIF-5A from HeLa Cells. Biochemistry, 34(45), 14693-14702. doi:10.1021/bi00045a010Łebska, M., Ciesielski, A., Szymona, L., Godecka, L., Lewandowska-Gnatowska, E., Szczegielniak, J., & Muszyńska, G. (2009). Phosphorylation of Maize Eukaryotic Translation Initiation Factor 5A (eIF5A) by Casein Kinase 2. Journal of Biological Chemistry, 285(9), 6217-6226. doi:10.1074/jbc.m109.018770Lee, S. B., Park, J. H., Kaevel, J., Sramkova, M., Weigert, R., & Park, M. H. (2009). The effect of hypusine modification on the intracellular localization of eIF5A. Biochemical and Biophysical Research Communications, 383(4), 497-502. doi:10.1016/j.bbrc.2009.04.049Li, C. H., Ohn, T., Ivanov, P., Tisdale, S., & Anderson, P. (2010). eIF5A Promotes Translation Elongation, Polysome Disassembly and Stress Granule Assembly. PLoS ONE, 5(4), e9942. doi:10.1371/journal.pone.0009942Liu, Z., Duguay, J., Ma, F., Wang, T.-W., Tshin, R., Hopkins, M. T., … Thompson, J. E. (2008). Modulation of eIF5A1 expression alters xylem abundance in Arabidopsis thaliana. Journal of Experimental Botany, 59(4), 939-950. doi:10.1093/jxb/ern017MA, F., LIU, Z., WANG, T.-W., HOPKINS, M. T., PETERSON, C. A., & THOMPSON, J. E. (2010). Arabidopsis eIF5A3 influences growth and the response to osmotic and nutrient stress. Plant, Cell & Environment, 33(10), 1682-1696. doi:10.1111/j.1365-3040.2010.02173.xMaier, B., Ogihara, T., Trace, A. P., Tersey, S. A., Robbins, R. D., Chakrabarti, S. K., … Mirmira, R. G. (2010). The unique hypusine modification of eIF5A promotes islet β cell inflammation and dysfunction in mice. Journal of Clinical Investigation, 120(6), 2156-2170. doi:10.1172/jci38924Mandal, S., Mandal, A., Johansson, H. E., Orjalo, A. V., & Park, M. H. (2013). Depletion of cellular polyamines, spermidine and spermine, causes a total arrest in translation and growth in mammalian cells. Proceedings of the National Academy of Sciences, 110(6), 2169-2174. doi:10.1073/pnas.1219002110Moreno-Romero, J., Carme Espunya, M., Platara, M., Ariño, J., & Carmen Martínez, M. (2008). A role for protein kinase CK2 in plant development: evidence obtained using a dominant-negative mutant. The Plant Journal, 55(1), 118-130. doi:10.1111/j.1365-313x.2008.03494.xNishimura, K., Lee, S. B., Park, J. H., & Park, M. H. (2011). Essential role of eIF5A-1 and deoxyhypusine synthase in mouse embryonic development. Amino Acids, 42(2-3), 703-710. doi:10.1007/s00726-011-0986-zNISHIMURA, K., OHKI, Y., FUKUCHI-SHIMOGORI, T., SAKATA, K., SAIGA, K., BEPPU, T., … IGARASHI, K. (2002). Inhibition of cell growth through inactivation of eukaryotic translation initiation factor 5A (eIF5A) by deoxyspergualin. Biochemical Journal, 363(3), 761. doi:10.1042/0264-6021:3630761Pagnussat, G. C. (2005). Genetic and molecular identification of genes required for female gametophyte development and function in Arabidopsis. Development, 132(3), 603-614. doi:10.1242/dev.01595Park, J. H., Dias, C. A. O., Lee, S. B., Valentini, S. R., Sokabe, M., Fraser, C. S., & Park, M. H. (2010). Production of active recombinant eIF5A: reconstitution in E.coli of eukaryotic hypusine modification of eIF5A by its coexpression with modifying enzymes. Protein Engineering Design and Selection, 24(3), 301-309. doi:10.1093/protein/gzq110Park, M. H. (2006). The Post-Translational Synthesis of a Polyamine-Derived Amino Acid, Hypusine, in the Eukaryotic Translation Initiation Factor 5A (eIF5A). The Journal of Biochemistry, 139(2), 161-169. doi:10.1093/jb/mvj034Park, M. H., Lee, Y. B., & Joe, Y. A. (1997). Hypusine Is Essential for Eukaryotic Cell Proliferation. Neurosignals, 6(3), 115-123. doi:10.1159/000109117Patel, P. H., Costa-Mattioli, M., Schulze, K. L., & Bellen, H. J. (2009). The Drosophila deoxyhypusine hydroxylase homologue nero and its target eIF5A are required for cell growth and the regulation of autophagy. The Journal of Cell Biology, 185(7), 1181-1194. doi:10.1083/jcb.200904161Ren, B., Chen, Q., Hong, S., Zhao, W., Feng, J., Feng, H., & Zuo, J. (2013). The Arabidopsis Eukaryotic Translation Initiation Factor eIF5A-2 Regulates Root Protoxylem Development by Modulating Cytokinin Signaling. The Plant Cell, 25(10), 3841-3857. doi:10.1105/tpc.113.116236Saez, A., Robert, N., Maktabi, M. H., Schroeder, J. 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Crystal Structure of Human Spermine Synthase: IMPLICATIONS OF SUBSTRATE BINDING AND CATALYTIC MECHANISM*S⃞♦

The crystal structures of two ternary complexes of human spermine synthase (EC 2.5.1.22), one with 5′-methylthioadenosine and spermidine and the other with 5′-methylthioadenosine and spermine, have been solved. They show that the enzyme is a dimer of two identical subunits. Each monomer has three domains: a C-terminal domain, which contains the active site and is similar in structure to spermidine synthase; a central domain made up of four β-strands; and an N-terminal domain with remarkable structural similarity to S-adenosylmethionine decarboxylase, the enzyme that forms the aminopropyl donor substrate. Dimerization occurs mainly through interactions between the N-terminal domains. Deletion of the N-terminal domain led to a complete loss of spermine synthase activity, suggesting that dimerization may be required for activity. The structures provide an outline of the active site and a plausible model for catalysis. The active site is similar to those of spermidine synthases but has a larger substrate-binding pocket able to accommodate longer substrates. Two residues (Asp201 and Asp276) that are conserved in aminopropyltransferases appear to play a key part in the catalytic mechanism, and this role was supported by the results of site-directed mutagenesis. The spermine synthase·5′-methylthioadenosine structure provides a plausible explanation for the potent inhibition of the reaction by this product and the stronger inhibition of spermine synthase compared with spermidine synthase. An analysis to trace possible evolutionary origins of spermine synthase is also described